[Evaluation of spoligotyping-smear sputum as an alternative and culture-independent methodology for Mycobacterium tuberculosis genotyping]. / Evaluación de spoligotyping a partir de baciloscopías como metodología alterna e independiente de cultivo para la genotipificación de Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) remains as the second cause of death by an infectious disease preceded by the acquired immune deficiency syndrome (AIDS). Currently, laboratory techniques and methodologies of diagnosis and drug susceptibility testing are constantly changing. Therefore, it has been recommended the introduction of rapid assays based on the amplification of nucleic acids test (NAAT's) through a polymerase chain reaction (PCR). Based on this principle, outstands spoligotyping - as a genotype and molecular epidemiology tool in tuberculosis - it is standardized to use isolated bacteria for the study of Mycobacterium genome through the amplification of 43 non-repetitive sequences, located at the direct repetitive region 1 (RD1). AIM: Evaluation of spoligotyping from acid fast staining smears as an independent option from bacterial isolation to characterize Mycobacterium tuberculosis by using sputum samples from TB patients from National Cardiopulmonary Institute in Tegucigalpa, Honduras. METHOD: Of 37 patients with positive culture (and smear microscopy) for M. tuberculosis, 50 expectoration samples were obtained. Microbiological and molecular tests were performed in respiratory samples containing mycobacterial DNA from sputum smears, concentrates and solid culture, for identification and genotype analysis by spoligotyping technique. RESULTS: Spoligotyping was positive in 37/37 of positive culture samples (S: 100%), in 36/37 (S: 97.3%) of smear-positive samples and in 6/10 (S: 60%) of concentrate samples sputum. The intensity of positive smear microscopy had a direct relationship with the sensitivity of spoligotyping. DISCUSSION: This study combined the potential of a molecular epidemiology tool to analyse DNA from sputum samples in smears acid fast staining, it visualizes diagnosis and genotyping platform in developing countries gathering innovation and high sensitivity in the hibridization of specific olignonucleotides from positive smears (P+, P++, P+++). However, the low specificity showed the need to improve better agreement among genetic patterns compared to the standardized bacterial isolation from M. tuberculosis strains.

Evaluación de spoligotyping a partir de baciloscopías como metodología alterna e independiente de cultivo para la genotipificación de Mycobacterium tuberculosis / Evaluation of spoligotyping-smear sputum as an alternative and culture-independent methodology for Mycobacterium tuberculosis genotyping

INTRODUCCIÓN: Tuberculosis (TBC) sigue siendo la segunda causa de muerte por una enfermedad infecto-contagiosa después del síndrome de inmunodeficiencia humana adquirida (SIDA). Actualmente, el escenario de técnicas y metodologías de laboratorio para la identificación y drogo-sensibilidad está cambiando gradualmente. Se han recomendado e introducido ensayos rápidos basados en la amplificación de ácidos nucleicos (NAAT's) que se desarrollan mediante la reacción de polimerasa en cadena (RPC). Bajo este principio, se destaca spoligotyping -una herramienta de genotipificación y epidemiología molecular en TBC- estandarizada a partir de aislados bacterianos, que permite el estudio del genoma de Mycobacterium mediante la amplificación de 43 secuencias cortas no repetitivas, localizadas en la región de repetición directa (RD1). OBJETIVO: Evaluación de spoligotyping a partir de baciloscopías, como una opción independiente de cultivo, para la caracterización de Mycobacterium tuberculosis a partir de muestras de esputo en pacientes del Instituto Nacional Cardiopulmonar de Tegucigalpa, Honduras. MÉTODO: De 37 pacientes con cultivo (y baciloscopía) positivos para M. tuberculosis, se obtuvieron 50 muestras de expectoración. Se realizó estudio microbiológico y molecular en muestras respiratorias conteniendo ADN de micobacterias, a partir de baciloscopías, concentrados y cultivo, para la identificación y análisis genotípico a través de la técnica de spoligotyping. RESULTADOS: El spoligotyping fue positivo en 37/37 de muestras de cultivo positivo (S: 100%), en 36/37 (S: 97,3%) de muestras con baciloscopía positiva y en 6/10 (S: 60%) de muestras de concentrado de esputo. La intensidad de la baciloscopía positiva tuvo una relación directa con la sensibilidad de spoligotyping. DISCUSIÓN: El fusionar el potencial de una herramienta útil en epidemiología molecular para analizar muestras de ADN proveniente de baciloscopías, visualiza una plataforma diagnóstica y genotípica para países en vías de desarrollo como una alternativa innovadora y altamente sensible en la hibridación de oligonucleótidos específicos a partir material genético en baciloscopías (P+, P++, P+++), pero requiere mejorar la concordancia entre patrones genéticos obtenidos, comparables con el uso estandarizado de aislados de cepas de M. tuberculosis.

BACKGROUND: Tuberculosis (TB) remains as the second cause of death by an infectious disease preceded by the acquired immune deficiency syndrome (AIDS). Currently, laboratory techniques and methodologies of diagnosis and drug susceptibility testing are constantly changing. Therefore, it has been recommended the introduction of rapid assays based on the amplification of nucleic acids test (NAAT's) through a polymerase chain reaction (PCR). Based on this principle, outstands spoligotyping - as a genotype and molecular epidemiology tool in tuberculosis - it is standardized to use isolated bacteria for the study of Mycobacterium genome through the amplification of 43 non-repetitive sequences, located at the direct repetitive region 1 (RD1). AIM Evaluation of spoligotyping from acid fast staining smears as an independent option from bacterial isolation to characterize Mycobacterium tuberculosis by using sputum samples from TB patients from National Cardiopulmonary Institute in Tegucigalpa, Honduras. METHOD: Of 37 patients with positive culture (and smear microscopy) for M. tuberculosis, 50 expectoration samples were obtained. Microbiological and molecular tests were performed in respiratory samples containing mycobacterial DNA from sputum smears, concentrates and solid culture, for identification and genotype analysis by spoligotyping technique. RESULTS: Spoligotyping was positive in 37/37 of positive culture samples (S: 100%), in 36/37 (S: 97.3%) of smear-positive samples and in 6/10 (S: 60%) of concentrate samples sputum. The intensity of positive smear microscopy had a direct relationship with the sensitivity of spoligotyping. DISCUSSION: This study combined the potential of a molecular epidemiology tool to analyse DNA from sputum samples in smears acid fast staining, it visualizes diagnosis and genotyping platform in developing countries gathering innovation and high sensitivity in the hibridization of specific olignonucleotides from positive smears (P+, P++, P+++). However, the low specificity showed the need to improve better agreement among genetic patterns compared to the standardized bacterial isolation from M. tuberculosis strains.

Difference in immune response in vaccinated and unvaccinated Swedish individuals after the 2009 influenza pandemic.

BACKGROUND: Previous exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009-2010. METHODS: Cellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test. RESULTS: Previous seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms. CONCLUSIONS: Strong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras.

BACKGROUND: A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. METHODS: Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). RESULTS: M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. CONCLUSIONS: The pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.